Epilepsy is a curable disease, but nearly one out of four patients develops seizures that are resistant to anti- epileptic medications. Seizures are neurological conditions characterized by abnormal brain waves and decreased inhibition. The fundamental determinant of inhibition in the brain is the protein KCC2, which dictates how key anti-epileptic drug targets work. Without KCC2 these targets would no longer function properly, and consequently, neither would the first- and second-line anti-epileptic drug therapies. KCC2 was only recently found to be severely diminished in the brains of people suffering from drug-resistant epilepsy. This proposal represents the first attempt to unequivocally identify the loss o this protein as the common feature of drug- resistant seizures. Given the fundamental role of this protein, it is vital that the proposed experiments are completed for the benefit of people suffering from this as yet incurable disorder. The project will consist of an array of molecular, electrophysiological, and genetic experiments to confirm the hypothesized role of KCC2 in animal models of drug-resistant seizures. The first project aim utilizes a new strategy to examine impaired KCC2 function on the molecular scale. This will demonstrate that a deficit in KCC2 function impedes inhibitory signaling between brain cells. The second project aim utilizes a model of drug-resistant seizures in animal brain tissue to directly demonstrate that the deficit in KCC2 function causes this disorder. This will be the first demonstration of this process in any model of drug-resistant seizures. The final project aim will directly address a longstanding unanswered question in medicine. Physicians have known for years that drug-resistant seizures develop with time, slowly and sometimes rapidly evolving from a state that can be treated into one that cannot. These experiments will be the first demonstration in a living animal brain that the progressive loss of KCC2 function underlies the development of these drug-resistant seizures. Such a finding would suggest that rescuing KCC2 function could restore the therapeutic effectiveness of current anti-epileptic drugs. The overarching research objective is to lay the foundation for immediate testing of a novel therapeutic strategy that targets KCC2 function to improve the quality of life of those suffering from drug-resistant seizures.